Creatine and/or creatinine compositions and related methods

ABSTRACT

The disclosure is directed to methods related to creatinine, for example, a method for its safe administration to a human, a method for using creatinine to improve the bioavailability of creatine, and a metn.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/367,209 filed Mar. 27, 2019, which claimspriority to and the benefit of U.S. Provisional Patent Application Ser.No. 62/648,870, filed on Mar. 27, 2018, and U.S. Provisional PatentApplication Ser. No. 62/650,594, filed on Mar. 30, 2018, the contents ofwhich are hereby incorporated entirely herein by reference.

BACKGROUND

Creatine (Cr) is an endogenous nutrient that occurs in various tissuesof mammals, for example, in liver, kidneys, muscular tissue, braintissue, and blood. It appears in a free state as well as in the form ofcreatine phosphate. Creatine phosphate (CrP) and creatine are allostericregulators of cell processes. Creatine enhances the energy tissuemetabolism by increasing the energy reserve of ATP in the muscle andnerve cells.

In a cell's mitochondria, creatine interacts reversibly with adenosinetriphosphate (ATP) through the action of a creatine kinase enzyme thatcatalyzes the formation of creatine phosphate and adenosine diphosphate(ADP). Upon consumption of ATP in a cell, a great amount of ADP isreleased, which leads to a transfer of ortho-phosphate from CrP to ADP,and the initial ratio between ATP and ADP remains. Due to the highaffinity of creatine kinase to ADP, this process continues until acreatine phosphate concentration falls below several tens micromolar.This interaction between creatine and ATP maintains the ATPconcentration at a constant level at the moments of intense ATPconsumption. While other processes exist for replenishing ATP, such asglycolysis or oxidative phosphorylation, these processes refill ATPnoticeably slower than the interaction between ATP and creatine.

CrP represents a reserve of macroergic phosphate for maintaining themembrane potential, activation of metabolites or contractive activity ofa cell. CrP maintains the ATP level during a period of increasing ofenergy consumption in a cell, for example, via restoring anortho-phosphate residue on ADP. Like glycogen, CrP is one of the basicsources of the high-energy phosphates transformation cycle and therebyparticipates in oxidative phosphorylation of glucose that providesliberation of energy necessary for the functionality of muscular tissuecells, including skeletal muscles and the cardiac muscle. Since CrPprovides for regeneration of ATP with a significant speed, an increaseof creatine amount in the muscles raises the muscles capacity of CrP,enhances the muscles workability, and increases the muscle bulk.

It has been shown that oral administration of creatine increases thetotal creatine content in an organism. In particular, administration ofcreatine monohydrate at dosages up to 30 g for a few days increases thetotal creatine content in skeletal muscles of a human subject by morethan 20%. These properties of creatine make the usage of creatinemonohydrate as a dietary supplement or food additive attractive,especially as an addition to the diet of an athlete. As described in thePublished International Patent Application WO 94/02127, creatinemonohydrate in a daily dose 15 g was administered for at least two daysfor increasing the muscle force. Nowadays creatine is also recommendedas a dietary supplement or food additive for elderly people andvegetarians, as these sections of the population have a tendency to havedecreased or low creatine level in their muscles.

Besides the use in the dietary supplement and food industry, creatineand creatine phosphate have wide applications in medicine. For example,creatine and creatine phosphate are recommended for the treatment ofnervous system diseases such as diabetic and toxic neuropathies,Alzheimer's disease, Parkinson's disease, and stroke, and alsodisturbances of metabolism such as hyperglycemia and diabetes mellitus(see U.S. Pat. Nos. 6,706,764 and 6,193,973). Oral administration ofcreatine has also been disclosed to be useful in the treatment ofcardiac insufficiency and respiratory failure (WO/EP97/06225) and ofasthma (U.S. Pat. No. 6,093,746). Additionally, creatine phosphate hasbeen disclosed as being useful for the treatment of cardiovasculardiseases and for the treatment of new-growth tissue (U.S. Pat. No.5,219,846).

The modes of delivering creatine has been limited. Ready-to-drink (RTD)formulations and sports drinks, such as Gatorade®, represent amultibillion-dollar market. However, in spite of energy drinks andliquid dietary supplement formulations being very popular amongconsumers (whether athletes or people seeking to be healthy), there hasnever been an RTD energy drink, sports drink, or liquid dietarysupplement that contains an effective dose of creatine available toconsumers. In the past, a “creatine serum” was sold claiming to be theworld's first stable liquid creatine preparation. However, test resultsshowed that the product contained less than 2% of the creatine claimed,the rest having been converted to creatinine (Dash and Sawhney, 2002).Creatine salts as well as effervescent forms have been deployed toincrease the stability of creatine in aqueous solutions but the resultshave been disappointing (Ganguly et al, 2003).

The reason for these past failures is because creatine is not stable inaquatic environments, and the rate of degradation increases withdecreasing pH. Most drinks have pH of 4.4 or less both to preventbacterial contamination and for taste purposes. Thus, it is notsurprising that to this date no liquid creatine formulation where thecreatine can remain stable at room or near room temperature for aprolonged period of time has existed. As such, there is a need fordeveloping compositions and methods that offer the benefits of a stableliquid creatine product.

SUMMARY

The disclosure relates to compositions and methods that ensure thestability of creatine. The disclosure also relates to compositions ofcreatine and creatinine where the creatinine enhances the concentrationof creatine, bioavailability of creatine, maximum plasma concentration(C_(max)) of creatine, or total plasma concentration of creatine overtime (in human subjects), for example, as evidenced by area under thecurve (AUC) of creatine in subjects. In some embodiments, thecomposition is a solid composition comprising a creatine compound suchas creatine nitrate and creatinine or a suitable creatinine compound. Insome aspects, the compositions described herein are dietary supplementsor dietary supplement formulations, for example, a nutraceutical drinkproduct, a liquid food product, or a fortified food for example.

In some aspects, the ratio of the creatine compound to creatinine or asuitable creatinine compound by weight in the composition is between23:1 and 1:9. In some aspects, the molar ratio of the creatine compoundto creatinine or a suitable creatinine compound in the composition isbetween 20:1 and 1:9, between 2:1 and 1:4, or between 3:1 and 1:3. In acertain embodiment, the molar ratio of the creatine compound tocreatinine or a suitable creatinine compound in the composition is1:1.7. In a certain embodiment, the composition comprises 5 g ofcreatine nitrate and 5 g of creatinine. In another embodiment, thecomposition comprises 3 g creatine nitrate and 3 g creatinine in about475 ml or 16 oz of liquid in a ready-to-drink sports supplementformulation. In still another embodiment, the composition comprises 1.5g creatine nitrate, 3.5 g creatine monohydrate, and 5 g creatinine. Inother aspects, the weight of the creatinine compound is 5% to 800% theweight of the creatine compound, for example, the weight of thecreatinine compound is between 50% and 200% of the weight of thecreatine compound. In another embodiment, the composition is in a liquidform made from mixing the solid composition with water or a water-basedcomposition. Thus, the liquid composition comprises a creatine compound(preferably creatine nitrate, for example), creatinine or a suitablecreatinine compound, and water. For example, the composition comprising1.5 g creatine nitrate, 3.5 g creatine monohydrate, and 5 g creatinineis dissolved in water or a water-based composition. In a preferredembodiment, the resulting liquid composition has a pH of 4.4 or less,for example between 4.2 and 4.4 or about 4.4. In some aspects, the totalvolume of the liquid composition is about 16 fluid ounces or about 450ml.

In some embodiments, the composition, whether liquid or solid, comprisesone or more additional components selected from the group consisting ofa carrier, an excipient, a binder, a colorant, a flavoring agent, apreservative, a buffer, and a diluent. In some aspects, the compositionsof the invention may be in a dosage form selected from the groupconsisting of: a capsule, a cachet, a pill, a tablet, an effervescenttablet, a powder, a granule, a pellet, a bead, a particle, a troche, alozenge, a gel, a liquid, a suspension, a solution, an elixir, and asyrup.

The compositions disclosed herein may be used as a food additive,nutraceutical, or dietary supplement, such as, for example, an additionto the diet of a healthy person, a patient, an athlete, and the like.These compositions may also be used in preparation of liquidformulations intended for use by patients where creatine supplementationwould be beneficial, such as with patients suffering from cerebralcreatine deficiency syndromes, chronic obstructive pulmonary disease(COPD), congestive heart failure (CHF), depression, diabetes,fibromyalgia, Huntington's disease, idiopathic inflammatory myopathies(polymyositis, dermatomyositis), Parkinson's disease, mitochondrialmyopathies, multiple sclerosis, muscle atrophy, muscle cramps, neonatalapnea, neurological trauma, Rett syndrome, gyrate atrophy of the choroidand retina, hereditary motor and sensory neuropathy, schizophrenia,spinal muscular atrophy, and surgical recovery, amyotrophic lateralsclerosis (ALS, also known as Lou Gehrig's disease), osteoarthritis,rheumatoid arthritis, McArdle disease, and various muscular dystrophies.Additionally, such compositions may also be used in either oral orparenteral nutrition. Furthermore, the compositions could also be usedtopically in liquid or semiliquid formulations such as creams,emulsions, serums, solutions, spirits, aerosols, gels and the like topromote skin health and prevent skin aging.

The disclosure is also directed to a method of stabilizing creatine in aliquid wherein the creatine content of the liquid composition after amonth of storage at room or near room temperature is at least 70% of theamount of creatine originally placed in the liquid. In other aspects atleast 90% or 95% of the original creatine placed in the liquid remainsafter storage of a month or 3 months or 6 months or a year. Stability ofat least 90% of the original amount of creatine is critical, because USPharmacopoeia formulation guidelines require that ingredients, such ascreatine, must have at least 90% of the amount stated in the label. Themethod may include: providing an amount of creatine; providing an amountof creatinine; dissolving the amount of creatine in water to produce aliquid composition; and adding the amount of creatinine to the liquidcomposition. The amount of creatine is provided from an acceptable formof creatine, including, an anhydrous form, a salt, a solvate, or ahydrate (for example, anhydrous creatine, creatine monohydrate, creatineformic acid solvate, or preferably creatine nitrate). The amount ofcreatinine is provided from an acceptable form of creatinine, including,an anhydrous form, a salt, a solvate, or a hydrate.

In certain implementations of the method, the amount of creatine and theamount of creatinine are combined in water to produce the liquidcomposition. Thus, in some aspects, the steps of dissolving the amountof creatine in water to produce a liquid composition and adding theamount of creatinine to the liquid composition consist of dissolving theamount of creatine and the amount of creatinine in water. Where theamount of creatine and the amount of creatinine are combined with waterat different times, the amount of creatinine is added to the liquidcomposition formed from dissolving the amount of creatine in water nomore than a day after the amount of creatine is dissolved in water.Accordingly, in some aspects, the amount of creatinine is dissolvedfirst, and there is no time limit for when the amount of creatine isadded to the liquid composition.

In some implementations of the methods, the weight of the amount ofcreatinine or salt or hydrate thereof is 5% to 800% the weight of theamount of creatine, for example, the weight of the amount of creatinineor salt or hydrate thereof is between 50% and 200%. In otherimplementations, the molar ratio of the amount of creatine to the amountcreatinine or salt or hydrate thereof is between 2:1 and 1:4 or between3:1 and 1:3, for example, in the case of creatine nitrate and creatinineabout, 1:1.7. In certain implementations, the amount of creatine nitrateis 5 g and the amount of creatinine is 5 g.

The disclosure also relates to methods of improving the solubility ofcreatine in water and to methods of producing a composition forparenteral administration or intravenous administration of creatine tohumans. In certain embodiments, the methods are directed to increasingthe creatine-stabilizing effect of creatinine in an aqueous solutioncomprising creatine. These methods comprise providing an aqueoussolution comprising an amount of creatine and an amount of creatinineand adding an amount of nitrate (NO₃ ⁻) to the aqueous solutioncomprising creatine. In certain embodiments, the methods are directed toenhancing conversion of creatinine to creatine in an aqueous solution.These methods comprise providing an aqueous solution comprising anamount of creatinine and adding to the aqueous solution an amount ofnitrate (NO₃ ⁻). In some implementations of these methods, the aqueoussolution further comprises an amount of creatine. The amount of creatinein the aqueous solution (or water) remains stable at a wide range ofstorage temperature, for example, between refrigeration and roomtemperature.

In some aspects of the methods of increasing the creatine-stabilizingeffect of creatinine in an aqueous solution comprising creatine and ofthe methods of enhancing conversion of creatinine to creatine in anaqueous solution, the molar ratio of the amount of nitrate (NO₃ ⁻) tothe amount of creatinine is selected from the group consisting of:between about 20:1 and about 1:9, between about 10:1 and about 1:1,between about 3:1 and about 1:3, and between about 2:1 and about 1:4. Insome implementations, molar ratio of the amount of creatine to theamount of nitrate (NO₃ ⁻) is selected from the group consisting of:between about 20:1 and about 1:9, between about 10:1 and about 1:1,between about 3:1 and about 1:3, and between about 2:1 and about 1:4. Incertain implementations, the molar ratio of the amount of creatine tothe amount of nitrate (NO₃ ⁻) is 1:1.

Methods of increasing the bioavailability of creatine and to counter thenegative effect of caffeine on creatine supplementation are alsodisclosed. In one aspect, the method of improving the bioavailability ofcreatine comprises co-administering to a human subject desiring thebenefits of creatine supplementation with an effective amount ofcreatine, creatinine, and nitrate (NO₃ ⁻). Methods of safelyadministering creatinine to a subject in need thereof, for example forfive days or longer, are also disclosed.

In some aspects of the method of increasing the bioavailability ofcreatine and of the method of safely administering creatinine to asubject in need thereof, the effective amount of creatine administeredis at least 500 mg, the effective amount of creatinine administered isat least 500 mg, and the effective amount of nitrate (NO₃ ⁻)administered is at least 50 mg nitrate (NO₃ ⁻). In otherimplementations, the effective amount of creatine administered is atleast 1000 mg, the effective amount of creatinine administered is atleast 1000 mg, and the effective amount of nitrate (NO₃ ⁻) administeredis at least 100 mg nitrate (NO₃ ⁻). In still other implementations, theeffective amount of creatine administered is at least 2 g, the effectiveamount of creatinine administered is at least 2 g, and the effectiveamount of nitrate (NO₃ ⁻) administered is at least 250 mg. In yetanother implementation, the effective amount of creatine administered isat least 2 g, the effective amount of creatinine administered is atleast 2 g, and the effective amount of nitrate (NO₃ ⁻) administered isat least 500 mg. In another implementation, the effective amount ofcreatine administered is at least 5 g, the effective amount ofcreatinine administered is at least 3 g, and the effective amount ofnitrate (NO₃ ⁻) administered is at least 1000 mg.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph tracking the change in the creatine and creatininecontent of an exemplary liquid composition of the disclosure stored atroom temperature (about 25° C.) over a period of 14 months.

FIG. 2 is a graph tracking the change in the creatine and creatininecontent of an exemplary liquid composition of the disclosure stored atroom temperature (about 25° C.) over a period of a year.

FIG. 3 is a graph tracking the change in pH and in the creatine,creatinine, and nitrate content of a liquid composition produced fromdissolving 5 g creatine nitrate and 4 g creatinine with 500 ml water.The liquid composition was stored at room temperature (about 25° C.).

FIG. 4 is a graph tracking the change in pH and in the creatine,creatinine, and nitrate content of a liquid composition produced fromdissolving 5 g creatine nitrate and 4 g creatinine with 500 ml water.The liquid composition was stored in refrigeration (2-8° C.).

FIG. 5 is a graph tracking the change in pH and in the creatine,creatinine, and nitrate content of a liquid composition produced fromdissolving 5 g creatine nitrate and 5 g creatinine with 500 ml water.The liquid composition was stored at room temperature (about 25° C.).

FIG. 6 is graph tracking the change in pH and in the creatine,creatinine, and nitrate content of a liquid composition produced fromdissolving 1.5 g creatine nitrate and 1 g creatinine with 500 ml of amulticomponent energy drink. The liquid composition was stored at roomtemperature (about 25° C.). At day 60, the pH of the half of thesolution was adjusted to 4.4 to study the effect of slightly less acidicpH on the levels of creatine and creatinine in the solution.

DETAILED DESCRIPTION

Detailed aspects and applications of the disclosure are described belowin the following drawings and detailed description of the technology.Unless specifically noted, it is intended that the words and phrases inthe specification and the claims be given their plain, ordinary, andaccustomed meaning to those of ordinary skill in the applicable arts.

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the disclosure. It will beunderstood, however, by those skilled in the relevant arts, thatimplementations of the technology disclosed herein may be practicedwithout these specific details. It should be noted that there are manydifferent and alternative configurations, devices and technologies towhich the disclosed technologies may be applied. The full scope of thetechnology disclosed herein is not limited to the examples that aredescribed below.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a step” includes reference to one or more of such steps.

As used herein, the term “about” refers to a deviation up to but notmore than 10% of the given value, for example a deviation of 10%, 7.5%,5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the given value.

As used herein, the term “dietary supplement” refers to an addition tothe human diet which is not a natural food, which has additionalbeneficial effects on the body unattainable by regular nutrition. Insome aspects, a dietary supplement is manufactured to be used over time,allowing for precise dosing. In some aspects, a dietary supplementincludes fortified food.

As used herein, the term “nutraceutical” refers to a dietary supplement,a dietary ingredient, a food additive, or a fortified food that provideshealth benefits, including preventing, treating, or curing a physical ormental condition.

As used herein, the term “dietary ingredient” refers to a dietarysubstance for use by man to supplement the diet by increasing totaldietary intake.

As used herein, the term “food additive” refers to a substance that is acomponent added to food.

As used herein, the term “fortified food” refers to food where itsnutritional and health value is increased (or fortified) by theadditional of a dietary supplement, dietary ingredients, or foodadditive.

As used herein, the term “room temperature” encompasses of a range oftemperatures between about 15° C. and about 27° C., for example, betweenabout 15° C. and about 25° C., between about 18° C. and about 22° C., orabout 20° C.

As used herein, the time period of “a day” refers to a period of between18 and 30 hours, for example, between 22 and 26 hours or about 24 hours.

As used herein, the term “effective amount” refers to an amount thatinduces a measurable or observable physiological change in a human. Forexample, in certain embodiments, an effective amount of creatininerefers to an amount of creatinine that increases the bioavailability ofcreatine or an amount that counteracts the inhibitory effect of caffeineon the ergogenic effects of creatine.

The present disclosure addresses the need for ensuring the stability ofcreatine in a solution, for example of water or other liquid orwater-based formulations.

Whereas solid creatine is stable, the presence of water leads tointramolecular cyclization that converts creatine to creatinine (seeScheme 1).

The rate of creatine degradation in solution is not dependent on theconcentration of creatine but on the pH of the solution. Generally, thelower the pH and higher the temperature, the faster creatine becomescreatinine in solution (see, for example, Edgar and Shiver, 1925; Cannonet al., 1990; Dash et al., 2002). While creatine was relatively stablein solution at neutral pH (7.5 or 6.5), lowering of pH resulted in anincreased rate of degradation. After only three days of storage at 25°C., creatine degraded by 4% at pH 5.5, by 12% at pH 4.5, and by 21% atpH 3.5. Similarly, creatine monohydrate in solution stored at roomtemperature degraded into creatinine within several days, whilerefrigerating creatine monohydrate in solution slowed the rate ofdegradation (Ganguly et al., 2003). Accordingly, the rapid degradationof creatine in solution precludes the manufacture of shelf-stablebeverages containing efficacious amounts of the ingredient.

Another issue with creatine supplementation has been its limitedbioavailability and finding methods to improve its bioavailability aswell as its overall plasma levels. In the past it was erroneouslyassumed that creatine possesses a bioavailability of near 100% becauseof a 2007 article that assumed that since no creatine or creatinine wasdetected in feces, the bioavailability of creatine should be around 100%(Deldicque et al., 2008). However recent data from a radiokineticbioavailability study (a standard for pharmacokinetic studies thatproduces more detailed and accurate data than merely measuring thecontents of an orally administered compound excreted) indicated thatthis hypothesis is highly erroneous; in fact, creatine bioavailabilitywas shown to be sharply reduced with increasing doses (Alraddadi et al.,2018). This finding validated the hypothesis by McCall and Persky thatcreatine bioavailability is less than 100% because of bacterial floradegradation in the gastrointestinal tract, gastric degradation, sitedependent intestinal absorption and incomplete dissolution of creatinesolid dosage forms (McCall and Persky, 2007).

It was surprisingly discovered by the inventors that the presence ofcreatinine in a solution comprising creatine reduces the rate ofcreatine degradation, and in some cases, creatine degradation iseliminated. Therefore, the present disclosure is directed in part to aliquid composition (for example, a liquid food product or liquid dietarysupplement formulation) containing a creatine compound and a creatininecompound wherein the creatine is stable for at least one month whenstored at room temperature or near room temperature. The liquid alsopossesses increased stability, for example of creatine, duringrefrigerated storage.

The disclosure also relates to methods of stabilizing creatine in aliquid wherein the creatine content of the liquid composition after amonth, two months, or three or more months or over a year of storage atabout room temperature or no greater than room temperature is at least70% of the amount of creatine nitrate provided thus enabling thepreparation of a liquid dietary supplement formulation comprising stablecreatine. In some aspects, the methods of stabilizing creatine in aliquid results the amount of creatine in the liquid composition being atleast 90% or at least 95% of the amount of creatine nitrate providedafter a month, two months, or three or more months or over a year ofstorage at about room temperature or no greater than room temperature.The methods comprise providing an amount of creatine (for example,provided as a creatine compound selected from the group consisting ofanhydrous creatine and a salt or hydrate or solvate of creatine);providing an amount of creatinine (for example, provided as a creatininecompound selected from the group consisting of anhydrous creatinine anda salt or hydrate or solvate of creatinine); and dissolving the amountof creatine and/or the amount of creatinine in water or a water-basedcomposition. In some aspects, the water-based composition is aready-to-drink food product, dietary supplement, vegetable juice, orfruit juice.

In some implementations, the amount of creatine is first dissolved inwater or water-based composition to produce a liquid composition and theamount of creatinine is then added to the liquid composition. In apreferred implementation, the amount of creatinine is added to theliquid composition, preferably no more than a day after creatine isdissolved in water or water-based composition. However, creatinine maybe added to the liquid composition formed from dissolving creatine in aliquid more than a day after the creatine is dissolved. In suchimplementations, precise formulation and labeling for the resultingcomposition is difficult as creatine may have degraded a significantamount according to labeling regulations.

In other implementations, the amount of creatine and the amount ofcreatinine are both dissolved in water or water-based composition toproduce a liquid composition, which can also be the liquid food productor liquid dietary supplement formulation.

In still other implementations, the amount of creatinine is firstdissolved in water or water-based composition to produce a liquidcomposition, and the amount of creatine is then dissolved in the liquidcomposition at a later time. In such implementations, the timing of whencreatine is dissolved in the liquid composition is not important, as thedissolved creatinine does not lose its ability to stabilize creatine inwater over time.

In some implementations of the methods, the weight of the amount ofcreatinine or salt or hydrate thereof is 5%-800% or 50-200% the weightof the amount of creatine compound. In other implementations, the molarratio of the amount of the creatine compound to the amount of thecreatinine or salt or hydrate thereof is between about 23:1 and 1:9, forexample, between 20:1 and 1:9, between 2:1 and 1:3, between 3:1 and 1:3,1:1, or 1:1.7. In some embodiments, the creatine compound provides atleast 500 mg creatine, at least 1000 mg creatine, at least 2 g creatine,or at least 5 g creatine, while the amount of creatinine provided is atleast 500 mg, at least 1000 mg, at least 2 g, or at least 3 g. Incertain implementations, the amount of creatine nitrate is 5 g and theamount of anhydrous creatinine is 4 g. In another implementation, theamount of the creatine compound consists of 1.5 g creatine nitrate and3.5 g anhydrous creatine while the amount of creatinine compoundconsists of 5 g anhydrous creatinine. In another implementation, theamount of the creatine compound consists of 3 g creatine nitrate and 2 ganhydrous creatine while the amount of creatinine compound consists of 5g creatinine.

In some implementations of the methods for increasing the stability ofcreatine in solution, the methods further comprise providing at leastone source of nitrate (NO₃ ⁻), wherein the at least one source ofnitrate (NO₃ ⁻) is dissolved with the creatine compound and/or thecreatinine compound in water or water-based composition. In suchimplementations, the source of nitrate (NO₃ ⁻) enhances the conversionof creatinine to creatine in the solution. Thus, such methods increasethe creatine-stabilizing effect of creatinine in an aqueous solution andenhances conversion of creatinine to creatine in an aqueous solution.Accordingly, in some aspects, the disclosure is also directed to methodsof increasing the creatine-stabilizing effect of creatinine in anaqueous solution and to methods of enhancing the conversion ofcreatinine to creatine in an aqueous solution.

In some implementations, the amount of nitrate (NO₃ ⁻) provided by theat least one source of nitrate (NO₃ ⁻) results in a molar ratio with theamount of creatinine of between about 20:1 and about 1:9, between about10:1 and about 1:1, between about 3:1 and about 1:3, or between about2:1 and about 1:4. In some implementations, the amount of nitrate (NO₃⁻) provided by the at least one source of nitrate (NO₃ ⁻) results in amolar ratio with the amount of creatine of between about 20:1 and about1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3,or between about 2:1 and about 1:4. In certain embodiments, the molarratio of the amount of creatine to the amount of nitrate (NO₃ ⁻)provided by the at least one source of nitrate (NO₃ ⁻) is 1:1. In suchembodiments, the source of nitrate (NO₃ ⁻) may be creatine nitrate. Insome aspects, the at least one source of nitrate (NO₃ ⁻) provides atleast 50 mg nitrate (NO₃ ⁻), at least 100 mg nitrate (NO₃ ⁻), at least250 mg nitrate (NO₃ ⁻), at least 500 mg nitrate (NO₃ ⁻), at least 100 mgnitrate (NO₃ ⁻), between 50 mg and 1200 mg nitrate (NO₃ ⁻), between 60mg and 1200 mg nitrate (NO₃ ⁻), between 50 mg and 1500 mg nitrate (NO₃⁻), or between 60 mg and 1500 mg nitrate (NO₃ ⁻).

In some embodiments, the method further comprises adjusting the pH ofthe liquid composition (after the creatine compound and the creatininecompound are dissolved) to 4.4 or less, for example, between about 4.2and about 4.4. The pH can be adjusted using any acceptable pH buffer,for example, sodium hydroxide.

In some aspects, the disclosure also relates to a liquid compositioncomprising creatine and creatinine, for example a drink fortified withcreatine and creatinine, wherein the liquid composition comprises astable amount of creatine. The stable liquid creatine formulation isproduced by combining a creatine compound and creatinine compound into acomposition and then dissolving the composition in water or awater-based composition. In other implementations, either the creatinecompound or the creatinine compound is dissolved in water or water-basedcomposition before the other compound is dissolved. The order of whichof the creatine compound or the creatinine compound is dissolved firstin water or the water-based composition is not critical, thoughdissolved creatine should not be allowed to remain in water orwater-based composition alone for more than an hour. For example,creatine monohydrate can first be dissolved in 500 ml of water, and anhour later, creatinine is dissolved in the same solution. If thecreatinine compound is dissolved in water or water-based compositionfirst, there is no similar urgency for when the creatine compound isdissolved in the resulting solution. In some preferred implementations,the creatinine compound is first dissolved in water or water-basedcomposition. In some implementations, the liquid composition is producedby first mixing the creatine compound and the creatinine compoundseparately in water to produce two separate solutions and then mixingthe two solutions.

In some embodiments, the stable liquid creatine formulation furthercomprises reducing the water content of the composition or thickeningthe composition. Accordingly, in some aspects, the stable liquidcreatine formulation is semisolid, for example, in the form of anemulsion, a pudding, or a gel.

In another implementation, the creatine compound and the creatininecompound are dissolved in water or water-based composition separately toproduce a creatine solution and a creatinine solution before the twosolutions are combined to produce a liquid composition described herein.To ensure no significant degradation of creatine takes place (forexample, more than 90% of the creatine provided is degraded), the twosolutions are combined within a day, or preferably within one hourdissolving the creatine compound. In some aspects, the method furthercomprises thickening or reducing the moisture content of the creatinesolution and/or the creatinine solution, wherein combining the twosolutions produces a semisolid composition or semiliquid composition,for example, a gel or pudding. In other aspects, the method furthercomprises thickening or reducing the liquid composition to produce asemisolid composition, for example, a gel or pudding.

In some embodiments, the stable liquid creatine formulation has a pH of4.4 or less, for example, between 4.2 and 4.4 or preferably about 4.4.Accordingly, in some implementations, the method of producing the stableliquid creatine formulation further comprises buffering the solutioncontaining the dissolved creatine compound and the dissolved creatininecompound to a pH of 4.4 or less, for example, between 4.2 and 4.4 orabout 4.4.

In some aspects, the invention is also directed to solid compositionscomprising a creatine compound and creatinine compound. In some aspects,the weight of the creatinine compound is 5%-800% or 50-200% the weightof the creatine compound. In a preferred embodiment, the weight of thecreatinine provided by the creatinine compound is 5%-800% or 50% to 200%the weight of creatine provided by the creatine compound. In otherimplementations, the molar ratio of the creatine compound to thecreatinine compound is between about 23:1 and 1:9, for example, between20:1 and 1:9, between 2:1 and 1:3, between 3:1 and 1:3, 1:1, or 1:1.7.In some embodiments, the creatine compound provides at least 500 mgcreatine, at least 1000 mg creatine, at least 2 g creatine, or at least5 g creatine, while the amount of creatinine provided is at least 500mg, at least 1000 mg, at least 2 g, or at least 3 g. In one embodiment,the creatine compound in the composition is 5 g creatine nitrate and thecreatinine compound is 4 g anhydrous creatinine. In another embodiment,the amount of the creatine compound in the composition consists of 1.5 gcreatine nitrate and 3.5 g anhydrous creatine, while the amount ofcreatinine compound in the composition consists of 5 g anhydrouscreatinine. In still another embodiment, the amount of the creatinecompound in the composition consists of 3 g creatine nitrate and 2 ganhydrous creatine, while the amount of creatinine compound in thecomposition consists of 5 g creatinine.

In certain embodiments, the compositions including creatine andcreatinine are dietary supplements, for example, to increase the amountof creatine in one's diet. As such, in some aspects, the disclosure isalso directed to the use of creatinine as a dietary ingredient or as afood additive.

Prior to the present disclosure, creatinine was primarily considered awaste product from the normal breakdown of muscle tissue. As creatinineis produced, it is filtered through the kidneys and excreted in urine.To this day, no beneficial biological role for creatinine has beenestablished. In contrast, creatinine is believed to be a toxic compoundwhich can impair human performance and health. Tambaru et al. andGangopadhya et al. both describe creatinine as a compound which cancause kidney damage. In view of high levels of creatinine beingcorrelated with a bad health prognosis, such as high creatinine levelsin the urine indicating kidney failure, it would be unethical to studythe biological effects caused by extremely high levels of creatinine inhumans. While it would be unethical to administer extremely high levelsof creatinine in a human subject, animal studies have supported theavoidance of supplementing creatinine or taking action that results inhigh creatinine levels in the blood, tissues, or urine. In mice,administration of creatinine had a sedating or stupefying effect from aninjection of creatinine (see Lis and Bijan, 1970). A similar effect wasseen in dogs (Giovannetti et al., 1969). In addition to theirobservations of the animal's aberrant behavior, Giovannetti et al.further concluded that creatinine was also responsible for a significantdecrease in the animal's erythrocyte survival time. In human bloodcells, the addition of creatinine initiated a significant increase inspontaneous hemolysis. This same red cell lysing pattern was observed innormal human volunteers whom had ingested creatinine (Giovannetti etal., 1969). Barsotti's research in 1975 gave further evidence of thispotential membrane-associated molecular blockade by showing thatcreatinine was able to effectively inhibit glucose utilization byerythrocytes (Barsotti et al., 1975).

Often creatinine is found in creatine supplements, but due to theevidence suggesting extra creatinine would have deleterious effects,creatinine is considered an impurity in such compositions. Accordingly,strict regulations exist to limit the amount of creatinine in commercialcreatine powders, for example, Health Canada allows the import ofcreatine monohydrate powders that contain a maximum of 100 ppmcreatinine (0.01% or less by weight).

It was also surprisingly discovered that, in contrast to prior artdescribing creatinine as toxic, a waste product, useless, and harmful tohuman performance (athletic, mental, and otherwise), concomitantadministration of creatine and creatinine to human subjects actuallyyield beneficial effects. As shown in Example 7, concomitantadministration of creatine and creatinine resulted in improved creatinebioavailability, improved creatine maximum concentration, and improvedcreatine body utilization. Instead of hindering performance, creatinineactually increased the ergogenic effects of creatine without producingany toxic or performance inhibiting effects.

In some aspects, the disclosure relates to methods of increasing thebioavailability of creatine, the method comprising administering acreatine compound in combination with a creatinine compound. The methodalso results in greater serum concentration of creatine, greater muscleutilization of creatine, or overall beneficial effect of creatine. Asdemonstrated in Example 7, no negative effects are associated withco-administration of a creatine compound with a creatinine compound.Accordingly, the disclosure also relates to methods of safelyadministering creatinine to a subject in need thereof. In someimplementations, an amount of between 0.5 and 20 g creatine isadministered by the administration of the creatine compound and anamount of between 0.5 and 20 g creatinine is administered by theadministration of the creatinine compound. For example, at least 1.5 gcreatine, for example at least 2 g creatine is administered through theadministration of the creatine compound. In some implementations, atleast 1.5 g creatinine is administered through the administration of thecreatinine compound, for example when the amount of creatineadministered is at least 2 g.

In some aspects, the method of increasing the bioavailability ofcreatine further comprises administering an effective amount of a sourceof nitrate (NO₃ ⁻). Such methods also safely administered creatinine toa subject in need thereof. In some embodiments, the creatine compoundprovides at least 500 mg creatine, at least 1000 mg creatine, at least 2g creatine, or at least 5 g creatine; the amount of creatinine providedis at least 500 mg, at least 1000 mg, at least 2 g, or at least 3 g; andthe amount of nitrate (NO₃ ⁻) provided by the source of nitrate (NO₃ ⁻)is at least 50 mg, at least 100 mg, at least 250 mg, at least 500 mg, orat least 1000 mg. In certain embodiments, the amount of nitrate (NO₃ ⁻)provided by the source of nitrate (NO₃ ⁻) is between between 50 mg and1200 mg nitrate (NO₃ ⁻), between 60 mg and 1200 mg nitrate (NO₃ ⁻),between 50 mg and 1500 mg nitrate (NO₃ ⁻), or between 60 mg and 1500 mgnitrate (NO₃ ⁻).

As demonstrated in Example 9, Applicants also surprisingly discoveredthat solubility of creatine in water at standard temperature and ambientpressure can be increased without the need to reduce the pH of thesolution with the presence of creatinine. Accordingly, this disclosurealso relates to a method of increasing the solubility of creatine inwater. The method comprises adding a creatine compound to a water-basedcomposition comprising creatinine to create a creatine solution. Abenefit of this method is that the resulting composition comprisingdissolved creatine can have a pH of between about 7 and about 8, whichmakes the composition suitable for parenteral administration, such asintravenous administration. In some aspect, the water-based compositioncomprising creatinine is produced by dissolving a creatinine compound inwater or a water-based composition. In some implementations, the methodfurther comprises adjusting the pH of the creatine solution to a pH ofbetween about 7 and about 8. In some aspects, methods are also directedto method of producing a composition for parenteral or intravenousadministration of creatine to humans. In one implementation, the weightof the creatine in the water-based composition is 50 to 500% the weightof the creatine provided by the creatine compound.

It was also surprisingly discovered that co-administration of acreatinine compound with a creatine compound counteracted caffeine'sneutralizing effect on the ergogenic actions of creatine (see Example8). Thus, in some aspects, this disclosure also relates to a method ofneutralizing caffeine's negative effect on the ergogenic actions ofcreatine, where the method includes administering to a subject consumingcaffeine an effective amount of creatinine or a combination of aneffective amount of a creatine compound and an effective amount of acreatinine compound. For a subject consuming between 60 to 1200 mgcaffeine per day, the effective amount of creatine and creatinineadministered to ensure the effectiveness of dietary supplementation ofcreatine is between 1-30 g creatine per day and between 1-30 gcreatinine per day, for example, about 20 g creatine and about 20 gcreatinine per day. In certain implementations, the daily amount ofcreatine and creatinine is administered in multiple doses in a day, forexample split across two, three, or four doses.

In some aspects, the creatinine compound and/or the creatine compoundis/are administered to the subject consuming caffeine within a day ofthe consumption of caffeine. In some implementations, the creatinecompound and the creatinine compound are administered separately. Forexample, the creatinine compound is administered to the subject within aday, about 24 hours, or about 2 hours of the administration of thecreatine compound. In other implementations, the creatine compound andthe creatinine compound are administered in a dietary supplementcomposition comprising an effective amount of the creatine compound andan effective amount of the creatinine compound.

Thus, Applicants discovered that creatinine is suitable as a dietaryingredient or food additive. In view of creatinine's beneficialsupportive role in creatine supplementation, the disclosure also relatesto the use of creatinine in producing a food fortified with creatine. Assuch, the disclosure also relates to dietary supplements and fortifiedfoods comprising creatine.

In some implementations of the methods for neutralizing caffeine'sprohibitive effect on the ergogenic actions of creatine, the methodsfurther comprises administering to the subject consuming caffeine atleast one source of nitrate (NO₃ ⁻). In some aspects, the at least onesource of nitrate (NO₃ ⁻) provides at least 50 mg, at least 100 mg, atleast 250 mg, at least 500 mg, or at least 1000 mg. In certainembodiments, the amount of nitrate (NO₃ ⁻) provided by the source ofnitrate (NO₃ ⁻) is between 50 mg and 1200 mg nitrate (NO₃ ⁻), between 60mg and 1200 mg nitrate (NO₃ ⁻), between 50 mg and 1500 mg nitrate (NO₃⁻), or between 60 mg and 1500 mg nitrate (NO₃ ⁻).

In some implementations of using creatinine as a dietary ingredient orfood product, the dietary supplement or food product comprises at leastone source of nitrate (NO₃ ⁻). In some aspects, the at least one sourceof nitrate (NO₃ ⁻) provides at least 50 mg, at least 100 mg, at least250 mg, at least 500 mg, or at least 1000 mg. In certain embodiments,the amount of nitrate (NO₃ ⁻) provided by the source of nitrate (NO₃ ⁻)is between 50 mg and 1200 mg nitrate (NO₃ ⁻), between 60 mg and 1200 mgnitrate (NO₃ ⁻), between 50 mg and 1500 mg nitrate (NO₃ ⁻), or between60 mg and 1500 mg nitrate (NO₃ ⁻).

In some implementations of methods of producing a food fortified withcreatine, the methods further comprise adding to the food fortified withcreatine at least one source of nitrate (NO₃ ⁻). In other aspects, thefood fortified with creatine comprises at least one source of nitrate(NO₃ ⁻). In some aspects, the at least one source of nitrate (NO₃ ⁻)provides at least 50 mg, at least 100 mg, at least 250 mg at least 500mg, or at least 1000 mg. In certain embodiments, the amount of nitrate(NO₃ ⁻) provided by the source of nitrate (NO₃ ⁻) is between 50 mg and1200 mg nitrate (NO₃ ⁻), between 60 mg and 1200 mg nitrate (NO₃ ⁻),between 50 mg and 1500 mg nitrate (NO₃ ⁻), or between 60 mg and 1500 mgnitrate (NO₃ ⁻).

In some implementations of the methods of increasing the solubility ofcreatine in water, the methods further comprise adding at least onesource of nitrate (NO₃ ⁻) to the water-based composition comprisingcreatinine. In some aspects, the at least one source of nitrate (NO₃ ⁻)provides at least 50 mg, at least 100 mg, at least 250 mg, at least 500mg, or at least 1000 mg. In certain embodiments, the amount of nitrate(NO₃ ⁻) provided by the source of nitrate (NO₃ ⁻) is between 50 mg and1200 mg nitrate (NO₃ ⁻), between 60 mg and 1200 mg nitrate (NO₃ ⁻),between 50 mg and 1500 mg nitrate (NO₃ ⁻), or between 60 mg and 1500 mgnitrate (NO₃ ⁻).

The amount of creatine compound in the compositions of the invention(for both the solid composition and the liquid composition) is variabledepending on the desired supplemental amount of creatine. Generally, adose of creatine for supplementation includes amounts between 500 mg to25 g creatine per dose. However, the molar ratio of the creatinecompound and creatinine compound in the compositions of the inventionmay be between about 23:1 and about 1:9, for example, between about 20:1and about 1:3, between about 10:1 and about 1:1, between about 3:1 andabout 1:3, between about 2:1 and about 1:1, about 1:1 or about 1:1.7. Insome aspects, the amount of creatinine compound is between 5% and 800%(for example between 50% and 200%) the weight of creatine compound. Incertain embodiments, for example, where the dietary ingredients of thedietary supplement consist of a creatine compound and a creatininecompound, the ratio by weight of creatine (from the creatine compound)to creatinine (from the creatinine compound) is preferably 5.5-7 weightparts creatine to 8 weight parts creatinine. It is preferred that onlyminimal amount of the creatinine compound (lowest amount possible toproduce the desired effect, such as increased solubility orbioavailability of creatine or increased stability of creatine insolution) is included in the compositions of the invention. For example,in certain embodiments, the molar ratio of the creatine compound to thecreatinine compound is about 1:1.1 or about 1:1.7.

One exemplifying composition comprises about 5 g creatine nitrate (whichcorresponds to the composition providing about 3.34 g creatine) andabout 4 g creatinine. Another exemplifying composition comprises about 5g creatine nitrate and about 5 g anhydrous creatinine. Still anotherexemplifying composition comprises about 4 g creatine anhydrous andabout 5 g creatinine nitrate. In some aspects, the composition comprisesabout 4 g creatine and between about 4 g and about 5 g creatinine. Insome aspects, the amount of creatine in the solid composition isprovided as a composition consisting of 1.5 g creatine nitrate and 3.5 gcreatine monohydrate. In such composition, the amount of creatinine is 5g anhydrous creatinine. In another embodiment, the composition comprises5 g creatine nitrate is 5 g and 4 g anhydrous creatinine. In anotherimplementation, the composition comprises 3 g creatine nitrate, 2 ganhydrous creatine, and 5 g creatinine.

The corresponding liquid composition (for example, liquid food productor liquid dietary supplement formulation) would further comprise wateror some other water-based composition or liquid, such as a commercialsports drink formulation, to dissolve the creatine compound and thecreatinine compound. In some aspects, the amount of water or some otherwater-based composition or liquid is about 500 ml, about 475 ml, about16 fluid oz, or about 240 ml. In some embodiments, the liquidcomposition further comprises a pH buffer, wherein the pH buffer adjuststhe pH of the liquid composition to 4.4 or less, for example, betweenabout 4.2 and about 4.4. In certain embodiments, the pH of the liquidcomposition is about 4.4.

The concentration of creatine from the creatine compound in the liquidcomposition of the disclosure does not fall below 70%, preferably 90% or95%, of the original concentration of creatine during storage, forexample, at or around room temperature for at least a month, threemonths, six months, or a year. In some embodiments, the concentration ofcreatine in the liquid composition of the disclosure remains steady. Inparticular, the concentration of creatine after 30 days of storage atroom temperature remains the same or higher than the concentration ofcreatine on day 1. In some aspects, the concentration of creatine after30 days is higher than the concentration of creatine after 1 day.

Compositions and/or formulations of the present invention may be in anyform for administration, whether solid or liquid. For example, thecomposition and/or formulation is in the form of a capsule, a cachet, apill, a tablet, a powder, a granule, a pellet, a bead, a particle, atroche, a lozenge, a pastille, a solution, an elixir, a syrup, atincture, a suspension, an emulsion, a mouthwash, a spray, a drop, anointment, a cream, a gel, a paste, a transdermal patch, a suppository, apessary, cream, a gel, a paste, a foam, or combinations thereof forexample. It is convenient to have an efficacious dose of creatine in agood-tasting, already mixed drink. Thus, liquid compositions where thecreatine is stable at a greater than 95% amount over a long time (forexample, 30 days, a month, three months, six months, or a year) withoutrequiring refrigeration are preferred.

Compositions and/or formulations of the present invention may alsoinclude at least one additional ingredient.

In one aspect, the additional ingredient produces a composition withintermediate rigidity and/or intermediate fluidity properties betweensolid and liquid, which is described interchangeably herein as asemisolid composition, a semiliquid composition, or a quasi-solidcomposition. In such embodiments, the additional ingredient includes butis not limited to a semisolid lipophilic vehicle, a paste, asolubilizer, thickener, or a gelling agent. In some aspects, theadditional ingredient in a solid composition produces a semiliquidcomposition. In other aspects, the additional ingredient in a liquidcomposition produces a semisolid composition.

In some aspects, the at least one additional ingredient comprises anacceptable additive for human consumption. Accordingly, the at least oneadditional ingredient is at least one additive selected from the groupconsisting of: a solubilizer, an enzyme inhibiting agent, ananticoagulant, an antifoaming agent, an antioxidant, a coloring agent, acoolant, a cryoprotectant, a hydrogen bonding agent, a flavoring agent,a plasticizer, a preservative, a sweetener, and a thickener. Theseadditives may be solids or liquids, and the type of additive may begenerally chosen based on the type of administration being used. Thoseof ordinary skill in the art will be able to readily select suitableadditives from the disclosure in this document. In particularimplementations, the acceptable additive is a pharmaceuticallyacceptable. For example, pharmaceutically acceptable additives include,calcium phosphate, cellulose, stearic acid, croscarmellose cellulose,magnesium stearate, and silicon dioxide. In another aspect, the at leastone additional ingredient comprises an acceptable carrier for humanconsumption. Accordingly, the at least one additional ingredient is atleast one carrier selected from the group consisting of: an excipient, alubricant, a binder, a disintegrator, a diluent, an extender, a solvent,a suspending agent, a dissolution aid, an isotonization agent, abuffering agent, a soothing agent, and an amphipathic lipid deliverysystem. In some aspects, the at least one additional ingredient isselected from the group consisting of: a flavoring agent, a colorant, aviscosity modifier, a preservative, a fragrance, an amino acid, a saltof an amino acid, a vitamin, a mineral, a fatty acid, an enzyme, aco-enzyme, a mono-glyceride, a di-glyceride, a tri-glyceride ester oilsemulsifiers, a hydrolyzed protein, whey protein, a stabilizer, a flowmodifier, a chelating agent, an antioxidant, an anti-microbial, abenzoate, an alcohol, an ester of para-hydroxybenzoic acid, apropionate, and a surfactant.

In particular embodiment, the compositions and/or formulations of thepresent invention further comprise at least one source of nitrate (NO₃⁻). In some aspects, a source of nitrate is an inorganic nitrate salt(for example, sodium nitrate or potassium nitrate). In other aspects, asource of nitrate is a nitrate salt of an amino acid or a nitrate saltof an amino acid derivative, for example, the nitrate salt of arginine,agmatine, beta alanine, betaine, carnitine, creatine, citrulline,glutamine, L-histidine, isoleucine, leucine, norvaline, ornithine,valine, aspartic acid, cysteine, glycine, lysine, methionine,phenylalanine, proline, taurine, or tyrosine. Where the creatinecompound of the composition is creatine nitrate, the at least one sourceof nitrate in the composition does not include creatine nitrate. Instill other aspects, a source of nitrate is a botanical source, forexample juice, extract, powder, or other derivative product fromcabbage, spinach, beet leaf, beetroot, artichoke, asparagus, broad bean,eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato,summer squash, sweet potato, tomato, watermelon, broccoli, carrot,cauliflower, cucumber, pumpkin, chicory, dill, turnip, savoy cabbage,celeriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsley,celery, cress, chervil, lettuce, rocket (rucola), and other vegetablesor fruits known to containing high levels of nitrate. In preferredembodiments, the botanical source of nitrate is beet juice.

In certain embodiments, the at least one source of nitrate (NO₃ ⁻)provides between about 50 mg and about 2000 mg nitrate (NO₃ ⁻), forexample, between about 60 mg and 1200 mg nitrate (NO₃ ⁻).

Creatine Compound

The creatine compound includes anhydrous creatine or a salt, solvate, orhydrate of creatine. While the creatine compound may be any salt ofcreatine, it is preferable the creatine compound is creatine nitrate.Other creatine compounds for use in the disclosed compositions includesingle administration physiologically active salts, creatine'stautomeric, polymeric and/or isomeric forms, creatine's analog forms, orcreatine's derivative forms. It should be noted that as disclosedherein, the creatine compound does not include creatine esters andpeptides, such as creatine ethyl ester and creatinyl-L-leucine. Creatineesters and peptides are unsuitable for the compositions described hereinalthough they are generally stable in an acidic environment. Creatineesters and peptides are not actual sources of creatine, because cleavageof the peptide bond results in the formation of creatinine instead ofcreatine. Also in many cases creatine esters and peptides may beexcreted unchanged to at least some degree.

As a non-limiting example, the creatine compound may be selected fromthe group consisting of: creatine nitrate, creatine anhydrous, creatinemonohydrate, creatine hydrochloride, creatine acetate, creatine malate,creatine ascorbate, creatine phosphate, creatine adipate, creatineaspartate, creatine caproate, creatine cinammate, creatine formate,creatine formic acid solvate, creatine fumarate, creatine gluconate,creatine glucuronate, creatine glycerophosphate, creatine glycolate,creatine lactate, creatine hydrobromide, creatine malonate, creatinemethanesulfonate, creatine oleate, creatine orotate, creatinenicotinate, creatine pyroglutamate, creatine pyruvate, creatinestearate, creatine tartrate, creatine succinate, creatine citrate,creatine ferulate, and creatine toluenesulfonate.

Creatine nitrate has been synthesized and patented by the applicants.The applicants found that creatine nitrate is more stable in aqueouscompositions than creatine monohydrate and buffered creatine(kre-alkalyn). In preferred embodiments, the creatine compound iscreatine nitrate.

The chemical stability of creatine nitrate (CN), creatine monohydrate(CM), and buffered creatine (BC) were examined under two differentstorage conditions: (1) 37° C. in pH 2.5 buffer and (2) 40° C. in pH 6.8buffer. A concentration of about 10 mg/ml of CN, CM and BC were preparedin both pH 2.5 and pH 6.8 buffer and stored in stability chambers inscrew capped bottles at 37° C. and 40° C., respectively. The degradationrate constants for CN, CM and BC at 37° C. in pH 2.5 buffer were0.075±0.001, 0.119±0.011, and 0.108±0.002 per day, respectively, whilethe degradation rate constants at 40° C. in pH 6.8 buffer were0.115±0.001, 0.015±0.001, and 0.013±0.002 per day, respectively. The pHof CN samples at 40° C. in pH 6.8 buffer changed from 2.83±0.01 to4.31±0.01 within a period of 12 days. The pH changes noticed at 37° C.in pH 2.5 buffer samples over the same period of time for CM, and BCwere 3.08±0.01 to 4.12±0.01 and 3.11±0.01 to 4.16±0.01, respectively. Nosignificant change in pH was observed for the rest of the samples. Nochange in the color and the clarity was noticed over 12 days.

All the creatine samples followed first order degradation kinetics underboth these experimental conditions. The degradation rate constants forCN was found to be higher at 40° C. in pH 6.8 buffer as compared to at37° C. in pH 2.5 buffer. However, both CM and BC showed a faster rate ofdegradation at 37° C. in pH 2.5 buffer than at 40° C. in pH 6.8 buffer.The major degradation product detected was creatinine. For CN theincrease in pH was higher at 40° C. in pH 6.8 buffer as compared to 37°C. in pH 2.5 buffer. However, opposite effect was noticed for both CMand BC.

When creatine nitrate is combined with creatinine before dissolving intoa solution, the concentration of creatine in the solution remainsconstant even after storage at around 25° C. for a long period of time,for example, at least a month (see Examples 1 and 2).

Creatinine Compound

The creatinine compound of the compositions of the disclosure isselected from any form of creatinine, including single administrationphysiologically active salts, solvates, or hydrates, creatinine'stautomeric, polymeric and/or isomeric forms, creatinine's analog forms,or creatinine's derivative forms. The creatinine compound includesanhydrous creatinine or a salt or hydrate of creatinine. The specifickind of creatinine compound used in the composition of the inventionaffects the stability of creatine. The salts of creatinine for use inthe composition include salts of creatinine formed using either anorganic acid or an inorganic acid, although the stability of creatinenitrate could be affected with every different creatinine salt chosen.Such salts include, but are not limited to: creatinine nitrate,creatinine hydrochloride, creatinine acetate, creatinine malate,creatinine ascorbate, creatinine phosphate, creatinine adipate,creatinine aspartate, creatinine caproate, creatinine cinammate,creatinine formate, creatinine fumarate, creatinine gluconate,creatinine glucuronate, creatinine glycerophosphate, creatinineglycolate, creatinine lactate, creatinine hydrobromide, creatininemalonate, creatinine methanesulfonate, creatinine oleate, creatinineorotate, creatinine nicotinate, creatinine pyroglutamate, creatininepyruvate, creatinine ferulate, creatinine citrate, creatinine stearate,creatinine tartrate, creatinine succinate, and creatininetoluenesulfonate, creatinine pyruvate.

Examples

The disclosure is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication are incorporated herein by reference in their entirety forall purposes.

1. Stability of Creatine in a Liquid Composition Comprising CreatineNitrate and Creatinine Over a Period of 14 Months: Storage at RoomTemperature (25° C.)

Creatine nitrate (5 g, equaling 25.5 mmol or 3.34 g creatine) wascombined with creatinine (4 g equaling 35.4 mmol creatinine) and thendissolved in 500 ml of water. The solution was left at room temperature(about 25° C.). Over the period of 14 months, the amount of creatine andcreatinine in ppm were measured (see Table 1 and FIG. 1).

TABLE 1 Analysis Date Creatinine (ppm) Creatine (ppm) 2017 Oct. 2710014.99 5244.27 2017 Nov. 3 10086.3 5417.66 2017 Nov. 10 9909.825342.83 2017 Nov. 14 9880.29 5443.84 2017 Nov. 28 9644.02 5422.60 2017Dec. 31 8969.06 6108.93 2018 Jan. 30 8723.39 6041.02 2018 Mar. 2 8282.806127.22 2018 Mar. 27 8183.38 6075.87 2018 Apr. 28 8557.34 6830.62 2018Jun. 1 8161.07 6647.78 2018 Jun. 30 8422.65 7029.07 2018 Sep. 8 8401.575611.32 2018 Oct. 11 8522.94 5319.45 2018 Dec. 6 8015.59 5559.72 2019Jan. 26 7812.69 5602.11

Contrary to the observations in the prior art regarding the variousforms of creatine converting to creatinine over time, the creatinecontent in the liquid creatine nitrate-creatinine composition has notreduced over time in the liquid formulation of the invention, therebycreating a unique stable creatine solution that may be used in foods,dietary supplements, and pharmaceutical preparations for example. Theamount of creatine at day 30 of the current invention is at a minimumthe same concentration, if not a higher concentration of creatine thanthe amount of creatine at day 1. In fact, in the surprising results ofthe original experiment, the creatine content in the liquid formulationof the invention actually increased from the initial creatineconcentration, as the solution comprising creatine nitrate as thecreatine compound and creatinine is stored for longer than a month atroom temperature.

2. Stability of Creatine in a Liquid Composition Comprising CreatineNitrate and Creatinine Over a Period of 12 Months: Storage at RoomTemperature (25° C.)

Creatine nitrate (5 g, equaling 25.5 mmol creatine or 3.34 g creatine)was combined with creatinine (4 g equaling 35.4 mmol creatinine) andthen dissolved in 500 ml of water. The solution was left at roomtemperature (about 25° C.). Surprisingly, the creatine content in theliquid increased from the initial creatine concentration as the liquidwas stored at room temperature for longer than a month (see Table 2 andFIG. 2).

TABLE 2 Analysis Date Creatinine (ppm) Creatine (ppm) 2018 Jan. 188837.51 5130.22 2018 Jan. 19 8710.34 5057.29 2018 Jan. 20 8804.515147.82 2018 Jan. 24 8818.74 5231.74 2018 Jan. 30 8638.97 5198.99 2018Mar. 2 8493.77 6065.45 2018 Mar. 27 8521.45 5768.23 2018 Apr. 28 8906.066562.30 2018 Jun. 1 8326.96 6914.92 2018 Jun. 30 8633.51 6039.40 2018Sep. 8 8666.33 6106.06 2018 Oct. 11 8640.62 5564.04 2018 Dec. 6 8724.835579.68 2019 Jan. 26 7926.65 5121.643. Stability of Creatine in a Liquid Composition Comprising CreatineNitrate and Creatinine Over a Period of 7 Months: Storage at RoomTemperature (25° C.)

In 500 ml of water at room temperature, 5 g creatine nitrate and 4 gcreatinine were added and creatine, creatinine, nitrate and pH levelswere assessed at the time point intervals indicated in the table below.Creatine content did not degrade, but actually increased, after 210 daysof storage (see Table 3 and FIG. 3).

TABLE 3 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) pH 06673 8182 3153 4.39 1 6356 7951 3134 4.35 7 6357 8066 3142 4.28 30 57708080 3142 4.28 60 6119 8293 3177 4.31 90 5420 8260 3060 4.35 120 61808130 3170 4.45 150 6430 7970 3180 4.58 210 6590 7920 3220 4.464. Stability of Creatine in a Liquid Composition Comprising CreatineNitrate and Creatinine Over a Period of 7 Months: Storage inRefrigeration (2-8° C.)

In 500 ml of water at a temperature of between 2-8° C., 5 g creatinenitrate and 4 g creatinine were added. The solution was stored inrefrigeration (2-8° C.). Creatine, creatinine, nitrate and pH levelswere assessed at the time point intervals indicated in Table 4 and FIG.4.

TABLE 4 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) pH 06647 8151 3189 4.39 1 6358 8097 3128 4.34 7 7024 8241 3304 4.28 30 63897799 3156 4.22 60 6748 7903 3157 4.17 90 6110 8070 3100 4.21 120 65607940 3140 4.34 150 6530 7940 3190 4.41 210 6780 7810 3180 4.35

Similar to the previous example, creatine levels remained stable duringthe whole 210 days regardless of refrigeration. This is very important.Previous approaches to the problem of creatine degradation tried to userefrigeration to slowdown creatine degradation, but it was surprisinglydiscovered that refrigeration, which carries a lot of drawbacks likeneed of a refrigerator, or other cooling device, increased costs,hurdles in transportation, etc., is not required to ensuring thestability of creatine a liquid composition in the invention disclosedherein.

5. Stability of Creatine in a Liquid Composition Comprising CreatineNitrate and Creatinine Over a Period of 7 Months: Storage inRefrigeration (2-8° C.)

In 500 ml of water at room temperature, 5 g creatine nitrate and 5 gcreatinine were added and creatine, creatinine, nitrate and pH levelswere assessed at the time point intervals indicated in Table 5 and FIG.5.

TABLE 5 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) pH 06630 10075 3179 4.63 1 6468 10010 3148 4.61 7 6852 9791 3194 4.51 306774 9251 2984 4.45 60 7240 9242 3148 4.41 90 6840 9370 3540 4.37 1207740 9250 3240 4.49 150 7420 8910 2870 4.53 210 7920 8630 3200 4.50

As can be seen in the table and in FIG. 5, creatine levels actuallyincreased while creatinine levels decreased. This is unprecedented: inan acidic environment of 4.4, which is well known to favor thedegradation of creatine to creatinine, the opposite occurred. Not onlywas creatine not degraded, the total creatine content in the compositionincreased. The increased creatine content may be due to the conversionof creatinine to creatine.

6. Stability of Creatine Added to a Commercial Energy Drink

Creatine nitrate and creatinine were dissolved in a multicomponentenergy drink (1.5 g creatine nitrate and 1 g creatinine added to 500 mlof the energy drink), and the changes in pH and creatine and creatininecontent were measured (See Table 6 and FIG. 6). After the addition ofcreatine nitrate and creatinine, the drink had a resulting pH of 3.71.Creatine continued to degrade through day 60, where 62% of the beginningcreatine content was seemingly lost. On day 60, the liquid was split inhalf to examine the influence of the pH in the stability of thecreatine-creatinine composition. In one half, the pH was adjusted to 4.4using a pH buffer. Increasing the pH resulted in increased creatinecontent despite the pH remaining at an acidic level. At day 210, 82% ofthe original creatine content was restored in the half of the solutionwith adjusted pH. Thus, maintaining the pH to about 4.4 is important forcreatine's stability even in the presence of creatinine.

TABLE 6 Creatine Creatinine Nitrate Analysis Day (ppm) (ppm) (ppm) pHDay 0 1972 2306 951 3.71 Day 1 1911 1965 878 3.71 Day 7 1597 2689 9963.69 Day 30 2240 2770 891 3.8 Day 60 769 3150 946 3.8 Day 90 632 3740984 3.88 Day 90, pH adjusted 713 3220 4.52 Day 120 708 2990 950 3.86 Day120, pH adjusted 1300 2480 4.45 Day 150 726 3230 915 3.86 Day 150, pHadjusted 1350 2590 4.36 Day 210 845 3170 944 3.82 Day 210, pH adjusted1620 2760 4.357. Human Study Comparing the Effects of Administering CreatineMonohydrate, Creatine Nitrate, and the Disclosed Composition

A human study was designed to evaluate the effects of combining creatineand creatinine for bioavailability and performance. Ten healthy humanvolunteers (aged 20-25 years) were used to evaluate and compare theeffects of administering 3 g creatine monohydrate (CrM), 3 g creatinenitrate (CN, providing about 2 g creatine) or a composition comprising 3g creatine nitrate and 3 g creatinine (CN—CRN).

Each human subject was administered CN, CrM, or CN—CRN with a glass ofwater with a washout period of 7 days among each experiment. Creatineserum levels were assessed at 0, 5, 30, 45, 60, 90, 120 minutes afteradministration of CN, CrM, or CN—CRN. The average peak serum creatineconcentrations at 60-min sampling interval were significantly higher inCN—CRN group (183.7±15.5 μmol/L), as compared to CN group (163.8±12.9μmol/L) and CrM group (118.6±12.9 μmol/L) (P<0.001). CN—CRN resulted ina more powerful rise in serum creatine levels comparing to either CN orCrM after single-dose intervention, as evaluated with the area under theconcentration-time curve calculation (701.1±62.1 (mol/L)×min vs.622.7±62.9 (mol/L)×min vs. 466.3±47.9 (mol/L)×min; P<0.001). It is ofgreat note that the much higher levels of serum creatine in the CN—CRNwere achieved with 33% less creatine than the creatine monohydrategroup. Accordingly, co-administration of creatine and creatininesignificantly improves serum creatine concentration in human subjects.

Based on muscle biopsies taken from the subject, higher creatine musclelevels were seen when the subjects were treated with CN—CRN.

Nine of the subjects did not report any negative side effects asmeasured by a side effect reporting questionnaire. However, one subjectreported gastrointestinal disturbances with all three treatments (CrM,CN, and CN—CRN).

Liver and kidney function as measured by ALT AST remained unchangedwhile GFR estimation showed a slight less than 10% clinicallyinsignificant reduction.

8. Creatinine Neutralizes Caffeine's Counteraction on the ErgogenicActions of Creatine

Vandenberghe et al. found that the ergogenic effect of creatine onmuscle was completely eliminated by caffeine intake (Vandenberghe etal., 1996). As Hespel et al.'s experiments showed, this might be due toopposite effect of caffeine and creatine on muscle relaxation time.However, Applicants discovered that co-administration of creatine withcreatinine eliminated the neutralizing effect of caffeine with respectof creatine's ergogenic actions on muscle.

A 35-year-old male subject (weight of 240 lb) ingested creatine withcreatinine supplement formulation for six days. During the period ofsupplementation, the subject was advised to abstain from creatine richfoods and caffeine sources. Specifically, the subject ingested a dose of5 g creatine nitrate and 5 g creatinine four times a day (total dailysupplementation of 20 g creatine nitrate and 20 g creatinine) for fivedays. On the fifth and sixth day, the subject also consumed 350 mgcaffeine in the morning alongside the morning dose of creatine andcreatinine.

Prior to supplementation the maximum weight the subject could push forthree knee extensions was 365 lb. On the morning of the sixth day ofsupplementation, the subject could push 380 lb for three kneeextensions. Thus, an increase in strength and endurance was observeddespite the co-administration of caffeine with creatine.

9. Creatinine Increases the Solubility of Creatine in Water

A common problem with creatine in the production of liquid supplementsis creatine's low solubility in water. Creatine has a solubility of 13.3g/l in water, or 13.3 mg/ml, in 25° C. While one option of increasingthe solubility of creatine in water is to reduce the pH of the solution,the cost of this approach is the reduced stability of creatine insolution. Applicants surprisingly found that creatinine, an alkalinesubstance, can increase creatine solubility of creatine even while itcauses the pH of the solution to increase. Thus, in a solution of 10 gcreatinine in one liter of water, the maximum solubility of creatine at25° C. in water increased to 15.8 mg/ml or 15.8 g/L, which is an 18%increase of creatine's solubility in water. The increased watersolubility of creatine in the presence of creatinine without the need toreduce the pH of the solution enables the manufacture of solutions withhigher concentration of creatine for use as an injectable or intravenoussolution, where the preferred pH range is between 7-8 (Lee et al.,2013).

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We claim:
 1. A method of increasing the creatine-stabilizing effect of creatinine in an aqueous solution comprising creatine, the method comprising: providing an aqueous solution comprising an amount of creatine and an amount of creatinine; and adding an amount of nitrate (NO₃ ⁻) to the aqueous solution comprising creatine.
 2. The method of claim 1, wherein the molar ratio of the amount of nitrate (NO₃ ⁻) to the amount of creatinine is selected from the group consisting of: between about 20:1 and about 1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3, and between about 2:1 and about 1:4.
 3. The method of claim 1, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO₃ ⁻) is selected from the group consisting of: between about 20:1 and about 1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3, and between about 2:1 and about 1:4.
 4. The method of claim 1, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO₃ ⁻) is 1:1.
 5. The method of claim 1, wherein the amount of creatine remains stable between storage at refrigeration and storage at room temperature.
 6. A method of enhancing conversion of creatinine to creatine in an aqueous solution, the method comprising: providing an aqueous solution comprising an amount of creatinine; and adding to the aqueous solution an amount of nitrate (NO₃ ⁻).
 7. The method of claim 6, wherein in the aqueous solution further comprises an amount of creatine.
 8. The method of claim 6, wherein the molar ratio of the amount of creatinine to the amount of nitrate (NO₃ ⁻) is selected from the group consisting of: between about 20:1 and about 1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3, and between about 2:1 and about 1:4.
 9. The method of claim 7, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO₃ ⁻) is selected from the group consisting of: between about 20:1 and about 1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3, and between about 2:1 and about 1:4.
 10. The method of claim 7, wherein the molar ratio of the amount of creatinine to the amount of nitrate (NO₃ ⁻) is 1.7:1. 